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Abstract:

Robust and reliable molecular diagnostic screening tools for early
detection of esophageal and gastrointestinal tract cancers and
pre-cancerous lesions, such as Barrett's Esophagus, and esophageal
adenocarcinoma are provided. Included in the invention is an array of
miRNA probes specific for identifying, diagnosing and prognosticating
esophageal and gastrointestinal tract cancers and pre-cancerous lesions
in subjects from blood or serum samples. A biochip comprising the array
as well as methods for its use are also provided.

Claims:

1. An array of oligonucleotide probes for identifying miRNAs in a sample,
comprising probes that each selectively bind a mature miRNA; and a
platform; wherein the probes are immobilized on the platform; wherein at
least two probes selectively bind a human miRNA selected from human
miRNAs comprising sequences of SEQ ID NOS: 1-7 and 10-14 or portions or
fragments thereof; or at least two probes are selected from probes
comprising sequences of SEQ ID NOS: 1-7 and 10-14 or portions or
fragments thereof.

2. The array of claim 1, and further comprising at least one
randomly-generated oligonucleotide probe sequence used as a negative
control; at least one oligonucleotide sequence derived from a
housekeeping gene, used as a negative control for total RNA degradation;
at least one randomly-generated sequence used as a positive control; and
a series of dilutions of at least one positive control sequence used as
saturation controls; wherein at least one positive control sequence is
positioned on the array to indicate orientation of the array.

4. A biochip comprising a solid substrate further comprising at least two
oligonucleotide probes of claim 3, which are capable of hybridizing to a
target sequence under stringent hybridization conditions and attached at
spatially defined address on the substrate.

5. A method of determining oncogenic, cancerous, premalignant or
metaplastic changes the esophagus or gastrointestinal tract of a
mammalian subject comprising: (a) extracting miRNA from a sample obtained
from a mammalian subject; (b) contacting the miRNA from (a) with the
array of claim 3; (c) performing an analysis using the array of b) to
determine expression of at least one miRNA obtained from the sample; and
(d) comparing the expression of at least two or more miRNA obtained from
the sample tissue with the expression of at least one miRNA obtained from
a control sample, wherein a detectable change in the expression of at
least two or more miRNA obtained from the sample compared to control is
indicative of oncogenic, cancerous, premalignant or metaplastic changes
in the gastrointestinal tract of a mammalian subject.

6. The method of claim 5, wherein the sample obtained from a mammalian
subject is selected from the group consisting of: blood, serum and
plasma.

7. A method of staging the oncogenic, cancerous, premalignant or
metaplastic changes in the esophagus or gastrointestinal tract of a
mammalian subject comprising: a) obtaining a sample from the subject; b)
contacting the RNA from (a) with the array of claim 3; c) determining the
amount of at least two miRNA selected from the group consisting of
hsa-miR-200a, hsa-miR-345, hsa-miR-373, hsa-miR-630, hsa-miR-663,
hsa-miR-765, hsa-miR-625, hsa-miR-93, hsa-miR-106b, hsa-miR-155,
hsa-miR-130b, hsa-miR-30a, hsa-miR-301a, hsa-miR-15b or portions or
fragments thereof of any of these miRNAs, or the amount of a precursor
molecule of the at least one miRNA in the sample from the subject; d)
comparing the amount of the at least two miRNA or the amount of a
precursor molecule of the at least two miRNA of a) with at least one or
more reference or control amounts; and wherein when a detectable change
in the amount of at least two microRNA obtained from the sample compared
to the reference or control, the stage of the oncogenic, cancerous,
premalignant or metaplastic changes in the gastrointestinal tract of a
mammalian subject is determined.

8. The method of claim 5 wherein when the amount of two or more miRNA
identified are increased over the amount of control miRNA, it is
indicative of the development of esophageal adenocarcinoma (EAC) or the
condition known as Barrett's esophagus (BE).

9. A method of determining oncogenic, cancerous, premalignant or
metaplastic changes the esophagus or gastrointestinal tract of a
mammalian subject comprising: (a) extracting RNA from a sample obtained
from a mammalian subject; (b) determining the expression of at least two
miRNA obtained from the sample; and (c) comparing the expression of at
least two miRNA obtained from the sample tissue with the expression of at
least one miRNA obtained from a control sample, wherein a detectable
change in the expression of at least one miRNA obtained from the sample
compared to control is indicative of oncogenic, cancerous, premalignant
or metaplastic changes in the gastrointestinal tract of a mammalian
subject.

11. The method of claim 10, wherein the oncogenic, cancerous,
premalignant or metaplastic changes are indicative of the development of
esophageal adenocarcinoma (EAC) or the condition known as Barrett's
esophagus (BE).

12. The method of claim 11, wherein the sample obtained from a mammalian
subject is selected from the group consisting of: blood, serum and
plasma.

13. A method of staging the oncogenic, cancerous, premalignant or
metaplastic changes in the esophagus or gastrointestinal tract of a
mammalian subject comprising: a) obtaining a sample from the subject; b)
determining the amount of at least two miRNA selected from the group
consisting of hsa-miR-200a, hsa-miR-345, hsa-miR-373, hsa-miR-630,
hsa-miR-663, hsa-miR-765, hsa-miR-625, hsa-miR-93, hsa-miR-106b,
hsa-miR-155, hsa-miR-130b, hsa-miR-30a, hsa-miR-301a, hsa-miR-15b or
portions or fragments thereof, or the amount of a precursor molecule of
the at least two miRNA in the sample from the subject; c) comparing the
amount of the at least two miRNA or the amount of a precursor molecule of
the at least two miRNA of a) with at least one or more reference or
control amounts; and wherein when a detectable change in the amount of at
least two miRNA obtained from the sample compared to the reference or
control, the stage of the oncogenic, cancerous, premalignant or
metaplastic changes in the gastrointestinal tract of a mammalian subject
is determined.

14. A method for diagnosing the progression the oncogenic, cancerous,
premalignant or metaplastic changes in the esophagus or gastrointestinal
tract of a mammalian subject comprising: a) obtaining a sample from the
subject; b) determining the amount of at least two miRNA selected from
the group consisting of hsa-miR-200a, hsa-miR-345, hsa-miR-373,
hsa-miR-630, hsa-miR-663, hsa-miR-765, hsa-miR-625, hsa-miR-93,
hsa-miR-106b, hsa-miR-155, hsa-miR-130b, hsa-miR-30a, hsa-miR-301a,
hsa-miR-15b or portions or fragments thereof, or the amount of a
precursor molecule of the at least two miRNA in the sample from the
subject; c) comparing the amount of the at least two miRNA or the amount
of a precursor molecule of the at least two miRNA of a) with at least one
or more reference or control amounts; and wherein when a detectable
change in the amount of at least two miRNA obtained from the sample
compared to the reference or control, the progression of the oncogenic,
cancerous, premalignant or metaplastic changes in the gastrointestinal
tract of a mammalian subject is determined.

15. (canceled)

Description:

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent
Application No. 61/468,194, filed on Mar. 28, 2011, which is hereby
incorporated by reference for all purposes as if fully set forth herein.

BACKGROUND OF THE INVENTION

[0003] As in the case of most diseases, in order to improve the prognosis
of patients, a diagnosis at an early stage is crucial. For example,
esophageal cancer (EC), the 8th-most common malignancy and 6th most
frequent cause of cancer death worldwide, exhibits highly aggressive
behavior. Barrett's esophagus (BE) is the obligate precursor lesion of
esophageal adenocarcinoma (EAC), one of the two major histologic subtypes
of EC. Early detection and close periodic surveillance of BE is the best
means to intervene in BE-associated neoplastic progression (BN). Existing
methods for detecting EC are endoscopic biopsy and histopathological
examinations, but they are limited due to their invasive nature and
inability to be applied in large-scale studies.

[0004] As many as 3 million Americans harbor BE; however, 40% or more of
EACs are diagnosed in subjects lacking any previous symptoms, and only 5%
of patients presenting with EAC carry an antecedent diagnosis of BE.
Early detection and close periodic surveillance of BE is the best means
to intervene in BE-associated neoplastic progression (BN). Nevertheless,
EAC develops in only 0.5%-1.0% of previously diagnosed BE patients
annually. Thus, most patients presenting with EAC have not benefited from
endoscopic (EGO) surveillance of BE. EGO is unsuitable and impractical
for population-based screening or detection of asymptomatic BN.
Furthermore, performing EGO based only on symptoms risks missing patients
with asymptomatic BE and/or EAC. Noninvasive diagnosis of BE would enroll
a higher proportion of individuals with BE into EGO surveillance programs
before they develop EAC, increasing BN diagnosis at earlier, more
survivable stages. At the same time, noninvasive diagnosis of EAC would
also improve outcome.

[0005] MicroRNAs (miRNAs or miRs) are short RNA oligonucleotides of
approximately 22 nucleotides that are involved in gene regulation.
MicroRNAs regulate gene expression by targeting mRNAs for cleavage or
translational repression. Although miRNAs are present in a wide range of
species including C. elegans, Drosophila and humans, they have only
recently been identified. More importantly, the role of miRNAs in the
development and progression of disease has only recently become
appreciated. Deregulated miRNA expression is implicated in onset and
progression of different diseases including, but not limited to embryonic
malformations and cancers.

[0006] As a result of their small size, miRNAs have been difficult to
identify using standard methodologies. A limited number of miRNAs have
been identified by extracting large quantities of RNA. MiRNAs have also
been identified that contribute to the presentation of visibly
discernable phenotypes. Expression array data shows that miRNAs are
expressed in different developmental stages or in different tissues. The
restriction of miRNAs to certain tissues or at limited developmental
stages indicates that the miRNAs identified to date are likely only a
small fraction of the total miRNAs.

[0007] Therefore, there still exists an imperative need to develop robust
and reliable molecular diagnostic screening tools for the early detection
of BE and/or EAC that will enhance the likelihood of cure and reduce the
incremental costs for the treatment of advanced disease.

SUMMARY OF THE INVENTION

[0008] In one or more embodiments, the present invention provides an array
of miRNA biomarkers that are detectable in the blood or serum of
subjects, which comprise a noninvasive diagnostic technology that is
sufficiently sensitive to detect oncogenic, cancerous, premalignant or
metaplastic changes in the gastrointestinal tract of a mammalian subject.

[0009] In accordance with an embodiment, the present invention provides an
array of oligonucleotide probes for identifying miRNAs, or portions or
fragments thereof, in a sample, comprising probes that each selectively
bind a mature miRNA, or a portion or fragment thereof, and a platform,
wherein the probes are immobilized on the platform, wherein at least two
probes selectively bind a human miRNA selected from human miRNAs
comprising sequences of SEQ ID NOS: 1-7 and 10-14, or portions or
fragments thereof, or at least two probes are selected from probes
comprising sequences of SEQ ID NOS: 1-7 and 10-14, or portions or
fragments thereof.

[0010] In accordance with another embodiment, the present invention
provides a biochip comprising a solid substrate, and further comprising
at least two oligonucleotide probes which selectively bind a human miRNA
selected from human miRNAs comprising sequences of SEQ ID NOS: 1-14, or
portions or fragments thereof, or at least two probes are selected from
probes comprising sequences of SEQ ID NOS: 1-14, or portions or fragments
thereof, which are capable of hybridizing to a target sequence under
stringent hybridization conditions and attached at spatially defined
address on the substrate.

[0011] In accordance with an further embodiment, the present invention
provides a method of determining oncogenic, cancerous, premalignant or
metaplastic changes the esophagus or gastrointestinal tract of a
mammalian subject comprising (a) extracting miRNA from a sample obtained
from a mammalian subject, (b) contacting the miRNA from (a) with the
array or the biochip as described above, (c) performing an analysis using
the array or biochip of b) to determine expression of at least one miRNA
obtained from the sample, and (d) comparing the expression of at least
two or more miRNA obtained from the sample tissue with the expression of
at least one miRNA obtained from a control sample, wherein a detectable
change in the expression of at least two or more miRNA obtained from the
sample compared to control is indicative of oncogenic, cancerous,
premalignant or metaplastic changes in the gastrointestinal tract of a
mammalian subject.

[0012] In accordance with still another embodiment, the present invention
provides a method of staging the oncogenic, cancerous, premalignant or
metaplastic changes in the esophagus or gastrointestinal tract of a
mammalian subject comprising a) obtaining a sample from the subject, b)
contacting the RNA from (a) with the array or biochip described above, c)
determining the amount of at least two miRNA selected from the group
consisting of hsa-miR-200a (SEQ ID NO: 1), hsa-miR-345 (SEQ ID NO: 2),
hsa-miR-373 (SEQ ID NO: 3), hsa-miR-630 (SEQ ID NO: 4), hsa-miR-663 (SEQ
ID NO: 5), hsa-miR-765 (SEQ ID NO: 6), hsa-miR-625 (SEQ ID NO: 7),
hsa-miR-93 (SEQ ID NO: 8), hsa-miR-106b (SEQ ID NO: 9), hsa-miR-155 (SEQ
ID NO: 10), hsa-miR-130b (SEQ ID NO: 11), hsa-miR-30a (SEQ ID NO: 12),
hsa-miR-301a (SEQ ID NO: 13), hsa-miR-15b (SEQ ID NO: 14), or portions or
fragments thereof, or the amount of a precursor molecule of the at least
one miRNA, or portions or fragments thereof, in the sample from the
subject, d) comparing the amount of the at least two miRNA or the amount
of a precursor molecule of the at least two miRNA of a) with at least one
or more reference or control amounts, and wherein when a detectable
change in the amount of at least two miRNA or portions or fragments
thereof, obtained from the sample compared to the reference or control,
the stage of the oncogenic, cancerous, premalignant or metaplastic
changes in the gastrointestinal tract of a mammalian subject is
determined.

[0013] In an embodiment, the present invention provides a method of
determining oncogenic, cancerous, premalignant or metaplastic changes the
esophagus or gastrointestinal tract of a mammalian subject comprising,
(a) extracting miRNA from a sample obtained from a mammalian subject, (b)
determining the expression of at least two miRNA obtained from the
sample, and (c) comparing the expression of at least two miRNA obtained
from the sample tissue with the expression of at least one miRNA obtained
from a control sample, wherein a detectable change in the expression of
at least one miRNA obtained from the sample compared to control is
indicative of oncogenic, cancerous, premalignant or metaplastic changes
in the gastrointestinal tract of a mammalian subject.

[0014] In accordance with another embodiment, the present invention
provides a method of staging the oncogenic, cancerous, premalignant or
metaplastic changes in the esophagus or gastrointestinal tract of a
mammalian subject comprising a) obtaining a sample from the subject, b)
determining the amount of at least two miRNA selected from the group
consisting of hsa-miR-200a, hsa-miR-345, hsa-miR-373, hsa-miR-630,
hsa-miR-663, hsa-miR-765, hsa-miR-625, hsa-miR-93, hsa-miR-106b,
hsa-miR-155, hsa-miR-130b, hsa-miR-30a, hsa-miR-301a, hsa-miR-15b, or
portions or fragments of any of these miRNAs thereof, or the amount of a
precursor molecule of the at least two miRNA in the sample from the
subject, c) comparing the amount of the at least two miRNA or the amount
of a precursor molecule of the at least two miRNA of a) with at least one
or more reference or control amounts, and wherein when a detectable
change in the amount of at least two miRNA obtained from the sample
compared to the reference or control, the stage of the oncogenic,
cancerous, premalignant or metaplastic changes in the gastrointestinal
tract of a mammalian subject is determined.

[0015] In accordance with a further embodiment, the present invention
provides a method for diagnosing the progression the oncogenic,
cancerous, premalignant or metaplastic changes in the esophagus or
gastrointestinal tract of a mammalian subject comprising a) obtaining a
sample from the subject, b) determining the amount of at least two miRNA
selected from the group consisting of hsa-miR-200a, hsa-miR-345,
hsa-miR-373, hsa-miR-630, hsa-miR-663, hsa-miR-765, hsa-miR-625,
hsa-miR-93, hsa-miR-106b, hsa-miR-155, hsa-miR-130b, hsa-miR-30a,
hsa-miR-301a, hsa-miR-15b or portions or fragments of any of these miRNAs
thereof, or the amount of a precursor molecule of the at least two miRNA
in the sample from the subject, c) comparing the amount of the at least
two miRNA or the amount of a precursor molecule of the at least two miRNA
of a) with at least one or more reference or control amounts, and wherein
when a detectable change in the amount of at least two miRNA obtained
from the sample compared to the reference or control, the progression of
the oncogenic, cancerous, premalignant or metaplastic changes in the
gastrointestinal tract of a mammalian subject is determined.

[0016] In accordance with yet another embodiment, the present invention
provides a use of at least two miRNA selected from the group consisting
hsa-miR-200a, hsa-miR-345, hsa-miR-373, hsa-miR-630, hsa-miR-663,
hsa-miR-765, hsa-miR-625, hsa-miR-93, hsa-miR-106b, hsa-miR-155,
hsa-miR-130b, hsa-miR-30a, hsa-miR-301a, hsa-miR-15b or portions or
fragments of any of these miRNAs thereof, or of a precursor molecule
thereof in a sample from a subject suffering from oncogenic, cancerous,
premalignant or metaplastic changes in the gastrointestinal tract for
identifying a subject being susceptible to gastrointestinal cancer
therapy.

BRIEF DESCRIPTION OF THE DRAWINGS

[0017] FIG. 1 is a table of miR-array data generated from human samples
that was normalized by the array control small RNA called Hurs.

[0018] FIG. 2 is a table of miR-array data generated from human samples
that was normalized by Agilent's GeneSpring GX 11.5 software.

[0019] FIG. 3 is a table of miR-array data generated from cell line
samples that was normalized by the array control small RNA called Hurs.

DETAILED DESCRIPTION OF THE INVENTION

[0020] By "nucleic acid" as used herein includes "polynucleotide,"
"oligonucleotide," and "nucleic acid molecule," and generally means a
polymer of DNA or RNA, which can be single-stranded or double-stranded,
synthesized or obtained (e.g., isolated and/or purified) from natural
sources, which can contain natural, non-natural or altered nucleotides,
and which can contain a natural, non-natural or altered internucleotide
linkage, such as a phosphoroamidate linkage or a phosphorothioate
linkage, instead of the phosphodiester found between the nucleotides of
an unmodified oligonucleotide. It is generally preferred that the nucleic
acid does not comprise any insertions, deletions, inversions, and/or
substitutions. However, it may be suitable in some instances, as
discussed herein, for the nucleic acid to comprise one or more
insertions, deletions, inversions, and/or substitutions.

[0021] In an embodiment, the nucleic acids of the invention are
recombinant. As used herein, the term "recombinant" refers to (i)
molecules that are constructed outside living cells by joining natural or
synthetic nucleic acid segments to nucleic acid molecules that can
replicate in a living cell, or (ii) molecules that result from the
replication of those described in (i) above. For purposes herein, the
replication can be in vitro replication or in vivo replication.

[0022] The nucleic acids used as primers in embodiments of the present
invention can be constructed based on chemical synthesis and/or enzymatic
ligation reactions using procedures known in the art. See, for example,
Sambrook et al. (eds.), Molecular Cloning, A Laboratory Manual, 3rd
Edition, Cold Spring Harbor Laboratory Press, New York (2001) and Ausubel
et al., Current Protocols in Molecular Biology, Greene Publishing
Associates and John Wiley & Sons, NY (1994). For example, a nucleic acid
can be chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of the
duplex formed upon hybridization (e.g., phosphorothioate derivatives and
acridine substituted nucleotides). Examples of modified nucleotides that
can be used to generate the nucleic acids include, but are not limited
to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil,
hypoxanthine, xanthine, 4-acetylcytosine, 5-(carboxyhydroxymethyl)
uracil, 5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine,
2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-substituted
adenine, 7-methylguanine, 5-methylaminomethyluracil,
5-methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine,
5'-methoxycarboxymethyluracil, 5-methoxyuracil,
2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v),
wybutoxosine, pseudouracil, queosine, 2-thiocytosine,
5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil,
uracil-5-oxyacetic acid methylester, 3-(3-amino-3-N-2-carboxypropyl)
uracil, and 2,6-diaminopurine. Alternatively, one or more of the nucleic
acids of the invention can be purchased from companies, such as
Macromolecular Resources (Fort Collins, Colo.) and Synthegen (Houston,
Tex.).

[0023] The nucleotide sequences used herein are those which hybridize
under stringent conditions preferably hybridizes under high stringency
conditions. By "high stringency conditions" is meant that the nucleotide
sequence specifically hybridizes to a target sequence (the nucleotide
sequence of any of the nucleic acids described herein) in an amount that
is detectably stronger than non-specific hybridization. High stringency
conditions include conditions which would distinguish a polynucleotide
with an exact complementary sequence, or one containing only a few
scattered mismatches from a random sequence that happened to have a few
small regions (e.g., 3-10 bases) that matched the nucleotide sequence.
Such small regions of complementarity are more easily melted than a
full-length complement of 14-17 or more bases, and high stringency
hybridization makes them easily distinguishable. Relatively high
stringency conditions would include, for example, low salt and/or high
temperature conditions, such as provided by about 0.02-0.1 M NaCl or the
equivalent, at temperatures of about 50-70° C.

[0024] The term "isolated and purified" as used herein means a protein
that is essentially free of association with other proteins or
polypeptides, e.g., as a naturally occurring protein that has been
separated from cellular and other contaminants by the use of antibodies
or other methods or as a purification product of a recombinant host cell
culture.

[0025] The term "biologically active" as used herein means an enzyme or
protein having structural, regulatory, or biochemical functions of a
naturally occurring molecule.

[0026] As used herein, the term "subject" refers to any mammal, including,
but not limited to, mammals of the order Rodentia, such as mice and
hamsters, and mammals of the order Logomorpha, such as rabbits. It is
preferred that the mammals are from the order Carnivora, including
Felines (cats) and Canines (dogs). It is more preferred that the mammals
are from the order Artiodactyla, including Bovines (cows) and Swines
(pigs) or of the order Perssodactyla, including Equines (horses). It is
most preferred that the mammals are of the order Primates, Ceboids, or
Simoids (monkeys) or of the order Anthropoids (humans and apes). An
especially preferred mammal is the human.

[0027] In accordance with one or more embodiments of the present
invention, it will be understood that the types of cancer diagnosis which
may be made, using the methods provided herein, is not necessarily
limited. For purposes herein, the cancer can be any cancer. As used
herein, the term "cancer" is meant any malignant growth or tumor caused
by abnormal and uncontrolled cell division that may spread to other parts
of the body through the lymphatic system or the blood stream.

[0028] The cancer can be a metastatic cancer or a non-metastatic (e.g.,
localized) cancer. As used herein, the term "metastatic cancer" refers to
a cancer in which cells of the cancer have metastasized, e.g., the cancer
is characterized by metastasis of a cancer cells. The metastasis can be
regional metastasis or distant metastasis, as described herein.

[0029] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or complete
treatment or prevention. Rather, there are varying degrees of treatment
or prevention of which one of ordinary skill in the art recognizes as
having a potential benefit or therapeutic effect. In this respect, the
inventive methods can provide any amount of any level of diagnosis,
staging, screening, or other patient management, including treatment or
prevention of cancer in a mammal Furthermore, the treatment or prevention
provided by the inventive method can include treatment or prevention of
one or more conditions or symptoms of the disease, e.g., cancer, being
treated or prevented. Also, for purposes herein, "prevention" can
encompass delaying the onset of the disease, or a symptom or condition
thereof.

[0030] "Complement" or "complementary" as used herein to refer to a
nucleic acid may mean Watson-Crick (e.g., A-T/U and C-G) or Hoogsteen
base pairing between nucleotides or nucleotide analogs of nucleic acid
molecules.

[0031] "Differential expression" may mean qualitative or quantitative
differences in the temporal and/or cellular gene expression patterns
within and among cells and tissue. Thus, a differentially expressed gene
may qualitatively have its expression altered, including an activation or
inactivation, in, e.g., normal versus disease tissue. Genes may be turned
on or turned off in a particular state, relative to another state thus
permitting comparison of two or more states. A qualitatively regulated
gene may exhibit an expression pattern within a state or cell type which
may be detectable by standard techniques. Some genes may be expressed in
one state or cell type, but not in both. Alternatively, the difference in
expression may be quantitative, e.g., in that expression is modulated,
either up-regulated, resulting in an increased amount of transcript, or
down-regulated, resulting in a decreased amount of transcript. The degree
to which expression differs need only be large enough to quantify via
standard characterization techniques such as expression arrays,
quantitative reverse transcriptase PCR, northern analysis, and RNase
protection.

[0032] "Identical" or "identity" as used herein in the context of two or
more nucleic acids or polypeptide sequences may mean that the sequences
have a specified percentage of residues that are the same over a
specified region. The percentage may be calculated by optimally aligning
the two sequences, comparing the two sequences over the specified region,
determining the number of positions at which the identical residue occurs
in both sequences to yield the number of matched positions, dividing the
number of matched positions by the total number of positions in the
specified region, and multiplying the result by 100 to yield the
percentage of sequence identity. In cases where the two sequences are of
different lengths or the alignment produces one or more staggered ends
and the specified region of comparison includes only a single sequence,
the residues of single sequence are included in the denominator but not
the numerator of the calculation. When comparing DNA and RNA, thymine (T)
and uracil (U) may be considered equivalent. Identity may be performed
manually or by using a computer sequence algorithm such as BLAST or BLAST
2.0.

[0033] "Probe" as used herein may mean an oligonucleotide capable of
binding to a target nucleic acid of complementary sequence through one or
more types of chemical bonds, usually through complementary base pairing,
usually through hydrogen bond formation. Probes may bind target sequences
lacking complete complementarity with the probe sequence depending upon
the stringency of the hybridization conditions. There may be any number
of base pair mismatches which will interfere with hybridization between
the target sequence and the single stranded nucleic acids described
herein. However, if the number of mutations is so great that no
hybridization can occur under even the least stringent of hybridization
conditions, the sequence is not a complementary target sequence. A probe
may be single stranded or partially single and partially double stranded.
The strandedness of the probe is dictated by the structure, composition,
and properties of the target sequence. Probes may be directly labeled or
indirectly labeled such as with biotin to which a streptavidin complex
may later bind.

[0035] "Substantially identical" used herein may mean that a first and
second sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%,
98% or 99% identical over a region of 8, 9, 10, 11, 12, 13, 14, 15, 16,
17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70,
75, 80, 85, 90, 95, 100 or more nucleotides or amino acids, or with
respect to nucleic acids, if the first sequence is substantially
complementary to the complement of the second sequence.

[0036] "Target" as used herein can mean an oligonucleotide or portions or
fragments thereof, which may be bound by one or more probes under
stringent hybridization conditions. "Target" as used herein may also mean
a specific miRNA or portions or fragments thereof, which may be bound by
one or more probes under stringent hybridization conditions.

[0038] In another embodiment, the present invention provides an array of
oligonucleotide probes for identifying miRNAs in a sample, comprising
probes that each selectively bind a mature miRNA, and a platform, wherein
the probes are immobilized on the platform, wherein at least three probes
selectively bind a human miRNAs consisting of sequences of SEQ ID NOS:
1-7 and 10-14; or at least three probes are selected from probes
consisting of sequences of SEQ ID NOS: 1-7 and 10-14. It will be
understood by those of ordinary skill that the array can bind any number
of oligonucleotide probes, including 4, 5, 6, 7, 8, 9, 10, 11, 12, 13,
and 14 probes at one time.

[0039] The nucleic acids of the present invention may also comprise a
sequence of a miRNA or a variant thereof. The miRNA sequence may comprise
from 13-33, 18-24 or 21-23 nucleotides. The miRNA may also comprise a
total of at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,
20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37,
38, 39, 40, 50, 60, 70, 80, 90 and up to 100 nucleotides. The sequence of
the miRNA may be the first 13-33 nucleotides of the pre-miRNA. The
sequence of the miRNA may also be the last 13-33 nucleotides of the
pre-miRNA. The sequence of the miRNA may comprise the sequence of SEQ ID
NOS: 1-14 or portions or fragments thereof.

[0040] A probe is also provided comprising a nucleic acid described
herein. Probes may be used for screening and diagnostic methods, as
outlined below. The probes may be attached or immobilized to a solid
substrate or apparatus, such as a biochip.

[0042] A biochip is also provided. The biochip is an apparatus which, in
certain embodiments, comprises a solid substrate comprising an attached
probe or plurality of probes described herein. The probes may be capable
of hybridizing to a target sequence under stringent hybridization
conditions. The probes may be attached at spatially defined address on
the substrate. More than one probe per target sequence may be used, with
either overlapping probes or probes to different sections of a particular
target sequence. In an embodiment, two or more probes per target sequence
are used. The probes may be capable of hybridizing to target sequences
associated with a single disorder.

[0043] The probes may be attached to the biochip in a wide variety of
ways, as will be appreciated by those in the art. The probes may either
be synthesized first, with subsequent attachment to the biochip, or may
be directly synthesized on the biochip.

[0044] The solid substrate may be a material that may be modified to
contain discrete individual sites appropriate for the attachment or
association of the probes and is amenable to at least one detection
method. Representative examples of substrates include glass and modified
or functionalized glass, plastics (including acrylics, polystyrene and
copolymers of styrene and other materials, polypropylene, polyethylene,
polybutylene, polyurethanes, Teflon, etc.), polysaccharides, nylon or
nitrocellulose, resins, silica or silica-based materials including
silicon and modified silicon, carbon, metals, inorganic glasses and
plastics. The substrates may allow optical detection without appreciably
fluorescing.

[0045] The substrate may be planar, although other configurations of
substrates may be used as well. For example, probes may be placed on the
inside surface of a tube, for flow-through sample analysis to minimize
sample volume. Similarly, the substrate may be flexible, such as a
flexible foam, including closed cell foams made of particular plastics.

[0046] The biochip and the probe may be derivatized with chemical
functional groups for subsequent attachment of the two. For example, the
biochip may be derivatized with a chemical functional group including,
but not limited to, amino groups, carboxyl groups, oxo groups or thiol
groups. Using these functional groups, the probes may be attached using
functional groups on the probes either directly or indirectly using a
linkers. The probes may be attached to the solid support by either the 5'
terminus, 3' terminus, or via an internal nucleotide.

[0047] The probe may also be attached to the solid support non-covalently.
For example, biotinylated oligonucleotides can be made, which may bind to
surfaces covalently coated with streptavidin, resulting in attachment.
Alternatively, probes may be synthesized on the surface using techniques
such as photopolymerization and photolithography.

[0048] A method of identifying a nucleic acid associated with a disease or
a pathological condition is also provided. The method comprises measuring
a level of the nucleic acid in a sample that is different than the level
of a control. In accordance with an embodiment, the nucleic acid is a
miRNA and the detection may be performed by contacting the sample with a
probe or biochip described herein and detecting the amount of
hybridization. PCR may be used to amplify nucleic acids in the sample,
which may provide higher sensitivity.

[0049] The level of the nucleic acid in the sample may also be compared to
a control cell (e.g., a normal cell) to determine whether the nucleic
acid is differentially expressed (e.g., overexpressed or underexpressed).
The ability to identify miRNAs that are differentially expressed in
pathological cells compared to a control can provide high-resolution,
high-sensitivity datasets which may be used in the areas of diagnostics,
prognostics, therapeutics, drug development, pharmacogenetics, biosensor
development, and other related areas.

[0050] The expression level of a disease-associated nucleic acid or miRNA
provides information in a number of ways. For example, a differential
expression of a disease-associated nucleic acid compared to a control may
be used as a diagnostic that a patient suffers from the disease.
Expression levels of a disease-associated nucleic acid may also be used
to monitor the treatment and disease state of a patient. Furthermore,
expression levels of a disease-associated miRNA may allow the screening
of drug candidates for altering a particular expression profile or
suppressing an expression profile associated with disease.

[0051] A target nucleic acid or portions or fragments thereof, may be
detected and levels of the target nucleic acid measured by contacting a
sample comprising the target nucleic acid with a biochip comprising an
attached probe sufficiently complementary to the target nucleic acid and
detecting hybridization to the probe above control levels.

[0052] The target nucleic acid or portions or fragments thereof, may also
be detected by immobilizing the nucleic acid to be examined on a solid
support such as nylon membranes and hybridizing a labeled probe with the
sample. Similarly, the target nucleic or portions or fragments thereof,
may also be detected by immobilizing the labeled probe to a solid support
and hybridizing a sample comprising a labeled target nucleic acid.
Following washing to remove the non-specific hybridization, the label may
be detected.

[0053] The target nucleic acid or portions or fragments thereof, may also
be detected in situ by contacting permeabilized cells or tissue samples
with a labeled probe to allow hybridization with the target nucleic acid.
Following washing to remove the non-specifically bound probe, the label
may be detected.

[0054] The detection of the target nucleic acid, or portions or fragments
thereof, can be through direct hybridization assays or can comprise
sandwich assays, which include the use of multiple probes, as is
generally known in the art.

[0055] A variety of hybridization conditions may be used, including high,
moderate and low stringency conditions as outlined above. The assays may
be performed under stringency conditions which allow hybridization of the
probe only to the target. Stringency can be controlled by altering a step
parameter that is a thermodynamic variable, including, but not limited
to, temperature, formamide concentration, salt concentration, chaotropic
salt concentration pH, or organic solvent concentration.

[0056] Hybridization reactions may be accomplished in a variety of ways.
Components of the reaction may be added simultaneously, or sequentially,
in different orders. In addition, the reaction may include a variety of
other reagents. These include salts, buffers, neutral proteins, e.g.,
albumin, detergents, etc. which may be used to facilitate optimal
hybridization and detection, and/or reduce non-specific or background
interactions. Reagents that otherwise improve the efficiency of the
assay, such as protease inhibitors, nuclease inhibitors and
anti-microbial agents may also be used as appropriate, depending on the
sample preparation methods and purity of the target.

[0057] A kit is also provided comprising an array of oligonucleotides as
described herein, or portions or fragments thereof, as well as a biochip
as described herein, along with any or all of the following: assay
reagents, buffers, probes and/or primers, and sterile saline or another
pharmaceutically acceptable emulsion and suspension base. In addition,
the kits may include instructional materials containing directions (e.g.,
protocols) for the practice of the methods described herein.

[0058] In accordance with another embodiment of the present invention, it
will be understood that the term "biological sample" or "biological
fluid" includes, but is not limited to, any quantity of a substance from
a living or formerly living patient or mammal Such substances include,
but are not limited to, blood, serum, plasma, urine, cells, organs,
tissues, bone, bone marrow, lymph, lymph nodes, synovial tissue,
chondrocytes, synovial macrophages, endothelial cells, and skin. In a
preferred embodiment, the fluid is blood or serum.

[0059] A method of diagnosis is also provided. The method comprises
detecting a differential expression level of two or more
disease-associated miRNAs in a biological sample. The sample may be
derived from a subject. Diagnosis of a disease state in a subject may
allow for prognosis and selection of therapeutic strategy. Further, the
developmental stage of cells may be classified by determining temporarily
expressed disease-associated miRNAs.

[0060] In situ hybridization of labeled probes to tissue arrays may be
performed. When comparing the levels of miRNA expression between an
individual and a standard, the skilled artisan can make a diagnosis, a
prognosis, or a prediction based on the findings. It is further
understood that the genes which indicate the diagnosis may differ from
those which indicate the prognosis and molecular profiling of the
condition of the cells may lead to distinctions between responsive or
refractory conditions or may be predictive of outcomes.

[0061] In accordance with an embodiment, the present invention provides an
array of oligonucleotide probes for identifying miRNAs in a sample,
comprising: probes that each selectively bind a mature miRNA; and a
platform, wherein the probes are immobilized on the platform; wherein at
least one probe selectively binds a human miRNA selected from human
miRNAs comprising sequences of SEQ ID NOS: 1-14 or a portion or fragment
thereof; or at least one probe is selected from probes comprising
sequences of SEQ ID NOS: 1-14 or a portion or fragment thereof.

[0062] Exemplary biochips of the present invention include an organized
assortment of oligonucleotide probes described above immobilized onto an
appropriate platform. Each probe selectively binds a miRNA in a sample.
In certain embodiments, each probe of the biochip selectively binds a
biologically active mature miRNA in a sample.

[0063] In accordance with another embodiment, the biochip of the present
invention can also include one or more positive or negative controls. For
example, oligonucleotides with randomized sequences can be used as
positive controls, indicating orientation of the biochip based on where
they are placed on the biochip, and providing controls for the detection
time of the biochip when it is used for detecting miRNAs in a sample.

[0064] Embodiments of the biochip can be made in the following manner. The
oligonucleotide probes to be included in the biochip are selected and
obtained. The probes can be selected, for example, based on a particular
subset of miRNAs of interest. The probes can be synthesized using methods
and materials known to those skilled in the art, or they can be
synthesized by and obtained from a commercial source, such as GeneScript
USA (Piscataway, N.J.).

[0065] Each discrete probe is then attached to an appropriate platform in
a discrete location, to provide an organized array of probes. Appropriate
platforms include membranes and glass slides. Appropriate membranes
include, for example, nylon membranes and nitrocellulose membranes. The
probes are attached to the platform using methods and materials known to
those skilled in the art. Briefly, the probes can be attached to the
platform by synthesizing the probes directly on the platform, or
probe-spotting using a contact or non-contact printing system.
Probe-spotting can be accomplished using any of several commercially
available systems, such as the GeneMachines® OmniGrid (San Carlos,
Calif.).

[0066] The miRNA sample can be amplified and labeled as is appropriate or
desired. If amplification is desired, methods known to those skilled in
the art can be applied. The miRNA samples can be labeled using various
methods known to those skilled in the art. In accordance with an
embodiment, the miRNA samples are labeled with digoxigenin using a
Digoxigenin (DIG) Nucleotide Tailing Kit (Roche Diagnostics Corporation,
Indianapolis, Ind.) in a GeneAmp® PCR System 9700 (Applied
Biosystems, Foster City, Calif.).

[0067] The labeled miRNA sample is incubated with the biochip, allowing
the miRNAs in the sample to hybridize with a probe specific for the
miRNAs in the sample. In certain embodiments, the labeled miRNA sample is
added to a DIG Easy Hyb Solution or Hybrid Easy Buffer (Roche Diagnostics
Corporation, Indianapolis, Ind.) that has been preheated to hybridization
temperature. The miRNA sample is the incubated with the biochip in the
solution, for example, for about 4 hours to about 24 hours.

[0068] The miRNAs in the sample can be detected, identified, and
quantified in the following manner. After the miRNA sample has been
incubated with the biochip for an appropriate time period, the biochip is
washed with a series of washing buffers, and then incubated with a
blocking buffer. When Digoxigenin (DIG) labeling of the miRNA samples has
been used, the biochip is then incubated with an Anti-DIG-AP antibody
(Roche Diagnostics Corporation, Indianapolis, Ind.). The biochip is them
washed with washing buffer and incubated with detection buffer, for
example, for about 5 minutes. NBT/BCIP dye
(5-Bromo-4-Chloro-3'-Indolyphosphate p-Toluidine Salt and NBT Nitro-Blue
Tetrazolium Chloride) diluted with detection buffer is added to the
biochip, which is allowed to develop in the dark, for example, for about
1 hour to about 2 days under humid conditions.

[0069] The biochips are scanned, for example, using an Epson Expression
1680 Scanner (Seiko Epson Corporation, Long Beach, Calif.) at a
resolution of about 1500 dpi and 16-bit grayscale. The biochip images are
analyzed using Array-Pro Analyzer (Media Cybernetics, Inc., Silver
Spring, Md.) software. Because the identity of the miRNA probes on the
biochip are known, the sample can be identified as including particular
miRNAs when spots of hybridized miRNAs-and-probes are visualized.
Additionally, the density of the spots can be obtained and used to
quantitate the identified miRNAs in the sample.

[0070] The identity and relative quantity of miRNAs in a sample can be
used to provide an miRNA profiles for a particular sample. An miRNA
profile for a sample includes information about the identities of miRNAs
contained in the sample, quantitative levels of miRNAs contained in the
sample, and/or changes in quantitative levels of miRNAs relative to
another sample. For example, an miRNA profile for a sample includes
information about the identities, quantitative levels, and/or changes in
quantitative levels of miRNAs associated a particular cellular type,
process, condition of interest, or other cellular state. Such information
can be used, for diagnostic purposes, drug development, drug screening
and/or drug efficacy testing. In an embodiment, the miRNAs of the present
invention are upregulated in subjects having pre-clinical EAC and BE. For
example, the presence of these miRNAs in high levels compared with
controls indicates a diagnosis of BE or EAC in a subject.

[0071] In another example, with regard to diagnostics, if it is known that
the presence or absence of a particular miRNA or group of miRNAs is
associated with the presence or absence of a particular condition of
interest, then a diagnosis of the condition can be made by obtaining the
miRNA profile of a sample taken from a patient being diagnosed.

EXAMPLES

[0072] Tissue Specimens. All patients provided written informed consent
under a protocol approved by the Institutional Review Boards at the
University of Maryland and Baltimore Veterans Affairs Medical Centers,
where all endoscopies were performed. Biopsies were taken using a
standardized biopsy protocol. Research tissues were obtained from
macroscopically apparent Barrett's epithelium or from mass lesions in
patients manifesting these changes at endoscopic examination, and
histology was confirmed using parallel aliquots from identical locations
obtained at the same endoscopy. All biopsy specimens were stored in
liquid nitrogen prior to DNA/RNA extraction.

[0073] miRNA extraction from serum. TRIzol LS Reagent (Invitrogen, cat.
no. 15596-018) was used to extract total RNA from sera of 16 patients
with EAC, BE or 12 age-matched normal EGD, and 16 tissues each of EAC, BE
or 12 age-matched normal EGD. 750 μl of TRIzol LS Reagent was added to
250 μl of serum sample and mixed thoroughly. After 5 minutes of
incubation, 200 μl of chloroform is added to the mixture, followed by
3 minutes of incubation. Then, the mixture was centrifuged at
12,000×g for 15 minutes at 4° C. After centrifugation, the
upper aqueous layer was transferred into new tubes, and 1.5 volumes of
100% ethanol was added to 1 volume of the aqueous layer. The mixture was
then added to RNeasy Mini kit (QIAGEN, cat. no. 74904) columns for the
total RNA extraction according to the manufacturer's instructions. 30
μl of RNase-free water was added onto the column to elute the RNA.

[0074] Quantitative RT-PCR (qRT-PCR) is an invaluable tool for highly
sensitive and accurate quantitation of miRNA expression, and constitutes
the standard method for independently validating microarray data. The
application of TaqMan (I RT-PCR technology permits the analysis of mature
miRNAs, rather than their precursors, ensuring the biological relevance
of miRNA expression.

[0075] Gene Expression Microarrays. Arrays containing 60-mer
oligonucleotide probes corresponding to 22,000 genes (Illumina HumanRef-8
Expression BeadChip v2, Illumina, San Diego, Calif.) were used to
construct an mRNA expression database for the cell lines studied. 100 ng
of total RNA was used for each labeling and hybridization reaction. Data
was normalized according to the LOWESS fitting curve method using MATLAB
(The MathWorks, Inc., Natick, Mass.).

[0076] MicroRNA Microarrays. MiRNA Labeling Reagent and Hybridization Kits
(Agilent, Santa Clara, Calif.) and Agilent's Human miRNA Microarray V1
which contains 471 human miRs, were used to generate global miR
expression profiles. This platform is designed to ensure extremely high
data fidelity and robustness. Each miR is represented by 30 probes on the
array (i.e., 15 replicates of 2 distinct probes hybridize to each miR).
Furthermore, these 30 probes are evenly distributed across the array to
minimize positional hybridization bias. 100 ng of total RNA from each
cell line was phosphatase-treated and then labeled with cyanine 3-pCp.
The labeled RNA was purified using Micro Bio-spin columns (BIO-RAD,
Hercules, Calif.) and subsequently hybridized to a human miR microarray
slide at 55° C. for 20 hours. After hybridization, the slides were
washed with Gene Expression Wash Buffer (Agilent) and scanned on an
Agilent Microarray Scanner (Agilent) using Agilent's Scan Control,
version A. 7.0.1 software. Data was collected and normalized to
non-functional small RNA internal controls.

[0078] Quantitative RT-PCR for miR Expression. TaqMan MicroRNA Assays,
Human (Applied Biosystems, Foster City, Calif.) were used to confirm miR
expression changes identified on miR microarrays, according to the
manufacturer's protocol. qRT-PCR was performed in triplicate. RNU6B
(RNU6B TaqMan microRNA Assay kit, Applied Biosystems) was used as an
internal control.

Example 1

[0079] MiR microarrays are hybridized to miRs extracted from matching
tissues and blood obtained from 16 subjects each with esophageal
adenocarcinoma (EAC), and compared to that of 12 healthy subjects.

[0080] In addition to these samples, miRs extracted from various normal
esophageal, Barrett's, and EAC cell lines (HEEPiC, CHTRT, GiHTRT, QHTRT,
and OE33 from ATCC, Manassas, Va.) were also used. For these experiments,
we used QIAGEN's miRNeasy Mini Kit for the actual miR extraction, and
Agilent's Human miRNA Microarray V1 which contains 471 human miRs.

Example 2

[0081] MiR-array data generated was normalized either by Agilent's
GeneSpring GX 11.5 software or by the array control small RNA called
Hurs. The normalized data was analyzed using significance analysis of
microarrays (SAM).

[0082] The serum data was first normalized using the Hurs array control
(FIG. 1). The top 144 highest fold-change overexpressed miRs were
selected that differed by a significant p-value between diseased and
normal control (NC). As a final filtering criterion, to ensure that serum
miRs will be robustly detectable, miRs were chosen whose individual serum
levels uniformly exceeded array background by at least a factor of 5.

[0084] The cell line data from various normal esophageal, Barrett's, and
EAC cell lines (HEEPiC, CHTRT, GiHTRT, QHTRT, and OE33) was processed in
the same way as the serum data in Example 2. The cell line data SAM
result generated 11 possible miR candidates (FIG. 3). We arrived at a
selection of 14 miR candidates (hsa-miR-200a, hsa-miR-345, hsa-miR-373*,
hsa-miR-630, hsa-miR-663, hsa-miR-765, hsa-miR-625, hsa-miR-93,
hsa-miR-106b, hsa-miR-155, hsa-miR-130b, hsa-miR-30a, hsa-miR-301a,
hsa-miR-15b) which commonly appeared or significant in 3 separate
analysis.

[0085] All references, including publications, patent applications, and
patents, cited herein are hereby incorporated by reference to the same
extent as if each reference were individually and specifically indicated
to be incorporated by reference and were set forth in its entirety
herein.

[0086] The use of the terms "a" and "an" and "the" and similar referents
in the context of describing the invention (especially in the context of
the following claims) are to be construed to cover both the singular and
the plural, unless otherwise indicated herein or clearly contradicted by
context. The terms "comprising," "having," "including," and "containing"
are to be construed as open-ended terms (i.e., meaning "including, but
not limited to,") unless otherwise noted. Recitation of ranges of values
herein are merely intended to serve as a shorthand method of referring
individually to each separate value falling within the range, unless
otherwise indicated herein, and each separate value is incorporated into
the specification as if it were individually recited herein. All methods
described herein can be performed in any suitable order unless otherwise
indicated herein or otherwise clearly contradicted by context. The use of
any and all examples, or exemplary language (e.g., "such as") provided
herein, is intended merely to better illuminate the invention and does
not pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of the
invention.

[0087] Preferred embodiments of this invention are described herein,
including the best mode known to the inventors for carrying out the
invention. Variations of those preferred embodiments may become apparent
to those of ordinary skill in the art upon reading the foregoing
description. The inventors expect skilled artisans to employ such
variations as appropriate, and the inventors intend for the invention to
be practiced otherwise than as specifically described herein.
Accordingly, this invention includes all modifications and equivalents of
the subject matter recited in the claims appended hereto as permitted by
applicable law. Moreover, any combination of the above-described elements
in all possible variations thereof is encompassed by the invention unless
otherwise indicated herein or otherwise clearly contradicted by context.